KR20180090558A - Novel microorganism with plastic-degrading activity and use thereof - Google Patents
Novel microorganism with plastic-degrading activity and use thereof Download PDFInfo
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09B—DISPOSAL OF SOLID WASTE NOT OTHERWISE PROVIDED FOR
- B09B3/00—Destroying solid waste or transforming solid waste into something useful or harmless
- B09B3/60—Biochemical treatment, e.g. by using enzymes
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09B—DISPOSAL OF SOLID WASTE NOT OTHERWISE PROVIDED FOR
- B09B3/00—Destroying solid waste or transforming solid waste into something useful or harmless
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- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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Abstract
Description
본 발명은 플라스틱 분해 활성을 갖는 신규 미생물 및 이의 용도에 관한 것으로서, 더 상세하게는 갈색거저리(Tenebrio molitor) 애벌레에서 분리되고 다양한 플라스틱을 선택적 또는 전체적으로 분해할 수 있는 신규 미생물 및 이를 이용한 플라스틱 전처리에 관한 것이다.The present invention relates to a novel microorganism having a plastics degrading activity and a use thereof, and more particularly to a microorganism having a plasticizing activity such as a brown goat ( Tenebrio The present invention relates to a novel microorganism capable of selectively or totally decomposing various plastics, and a plastic pretreatment using the microorganisms.
폐기물은 금속, 유리, 플라스틱 등 다양한 물질을 포함한다. 플라스틱은 산업 폐기물 및 생활 폐기물을 통틀어 금속보다 월등히 많은 양을 차지하고 있으며, 이에 따라 그 가치를 경제적으로 환산시켜보았을 때 금속을 뛰어넘는다. 한편, 플라스틱의 주원료인 석유의 고갈로 인해 폐기물로 배출되는 플라스틱의 재활용에 대한 필요성이 더욱 대두되고 있다. Waste includes various materials such as metals, glass, and plastics. Plastics account for much more than metals, both industrial and municipal waste, and thus leapfrog metals when they are converted economically. On the other hand, due to the depletion of petroleum, which is the main material of plastics, there is a growing need for the recycling of waste plastics.
플라스틱은 재활용 시 대개 한 제품 속에 다양한 종류의 플라스틱이 사용되고, 다양한 종류의 플라스틱들이 한꺼번에 수집되어 재활용된다. 이 때문에 각 플라스틱 재질은 타 플라스틱 재질이 불순물로 작용하여 단일 재질로서의 순도를 완벽히 유지하기 어려우며, 그 결과 품질이 떨어져 신재 플라스틱에 비해 경제적 가치가 약 34% 정도 낮게 평가된다. 특히 생활 폐기물 중에 섞여 있는 혼합 폐 플라스틱을 성상별로 경제성 있게 분리하기가 어려워 혼합 폐 플라스틱을 선별하는 기술 개발이 절실한 실정이다. 플라스틱 재활용 공정은 크게 전처리 과정 및 재생 과정으로 이루어지고, 전처리 과정에서 혼합 폐 플라스틱은 성상별로 분리 선별된다. 혼합 폐 플라스틱을 성상별로 분리 선별하기 위해 주로 습식 부유 선별 방법이 주로 사용되는데, 습식 부유 선별을 하기 위해서는 많은 양의 물이 필요하고 습식 부유 선별을 거치더라도 약 2% 정도의 타 재질이 섞여 있어서 저급의 플라스틱으로 재활용된다. 특히, PVC(Polyvinyl chloride)는 습식 부유 선별을 거친 플라스틱에 대해 이물질로 작용하므로 플라스틱 재활용 공정의 전처리 과정에서 PVC(Polyvinyl chloride)의 사전 제거가 필수적이다.When plastic is recycled, a wide variety of plastics are used in one product, and a wide variety of plastics are collected and recycled at the same time. For this reason, each plastic material is hard to maintain the purity as a single material because other plastic materials act as impurities. As a result, the quality is lowered and the economic value is estimated to be about 34% lower than that of the new plastic. Especially, it is difficult to economically separate blended waste plastics mixed in municipal wastes. The plastic recycling process consists largely of preprocessing and regeneration process. In the pretreatment process, the mixed waste plastic is separated and sorted according to the properties. A wet flotation screening method is mainly used to separate mixed waste plastics according to their characteristics. In order to select wet flotation, a large amount of water is required. Even if wet flotation is selected, about 2% of other materials are mixed, Of plastic. Particularly, PVC (polyvinyl chloride) acts as a foreign substance to the plastic which has been subjected to wet flotation, so it is necessary to remove the PVC (polyvinyl chloride) in the pretreatment process of the plastic recycling process.
한편, 최근 미생물을 이용하여 폐수나 폐기물에 함유된 플라스틱을 처리하는 기술이 개발되고 있다. 예를 들어, 대한민국 등록특허 제10-0350928호에는 호기성 조건에서 생육이 양호하며, 폴리비닐알코올의 분해능력이 개선된 신규 미생물 크레브시엘라 뉴모니에 씨제이-피브이에이 에이(Klebsiella pneumoniae CJ-PVA a; 기탁번호 제KFCC-11126호)와, 이를 이용하여 폴리비닐알코올이 함유된 폐수를 처리하는 방법이 개시되어 있다. 또한, 대한민국 등록특허 제10-0513931호에는 마이크로박테리움 바커리 엘씨 (Microbacterium barkeri LC) (기탁번호 : KCCM 10507)와 이를 이용하여 폴리비닐알코올을 생물학적으로 분해하는 방법이 개시되어 있다.On the other hand, a technique for treating plastics contained in wastewater or wastes using microorganisms has recently been developed. For example, Korean Patent No. 10-0350928 discloses a novel microorganism Crevicillinum mononitrate which has good growth under aerobic conditions and has improved ability to degrade polyvinyl alcohol. Klebsiella pneumoniae CJ- PVA a, deposit number KFCC-11126), and a method for treating wastewater containing polyvinyl alcohol using the same. Korean Patent No. 10-0513931 discloses a microbacterium barkeri LC (accession number: KCCM 10507) and a method for biologically degrading polyvinyl alcohol using the microbacterium barkeri LC.
갈색거저리(Tenebrio molitor)는 거저리과에 속하는 곤충의 일종이다. 갈색거저리의 애벌레는 흔히 밀웜(mealworm)이라 부르고 주로 애완동물의 먹이와 식용 곤충으로 많이 사용된다. 2015년에 밀웜(mealworm)이 폴리스티렌을 재사용이 가능한 유기물질로 분해할 수 있다는 사실이 발견되었다[Jordan, Rob. "Plastic-eating worms may offer solution to mounting waste, Stanford researchers discover". Stanford News Service. Stanford News Service. Retrieved 24 March 2016.]. 그러나 밀웜의 플라스틱 분해 메커니즘은 알려진바가 없고, 이로 인해 상업화도 시도되지 않았다.The brown duck ( Tenebrio molitor ) is a kind of insect belonging to the genus. The larvae of brown ducks are often called mealworms and are often used as food for pets and edible insects. In 2015, it was discovered that mealworm can break down polystyrene into reusable organic materials [Jordan, Rob. "Plastic-eating worms may offer solution for mounting waste, Stanford researchers discover". Stanford News Service. Stanford News Service. Retrieved 24 March 2016.]. However, there is no known mechanism for decomposing the plastic of the wheat worm, and commercialization has not been attempted.
본 발명은 종래의 기술적 배경하에서 도출된 것으로서, 본 발명의 목적은 다양한 플라스틱에 대해 분해 활성을 가지는 신규 미생물 및 이의 용도를 제공하는 데에 있다.The present invention has been made under the conventional technical background, and an object of the present invention is to provide a novel microorganism having decomposing activity against various plastics and its use.
본 발명의 발명자들은 갈색거저리 애벌레의 폴리스티렌 분해능력이 갈색거저리 애벌레의 장내에서 서식하는 미생물에 기인하다는 가정하에 갈색거저리 애벌레의 장내에서 서식하는 미생물을 분리하고, 분리된 미생물 중 일부가 다양한 플라스틱에 대해 우수한 분해 활성을 보인다는 점을 확인하고, 본 발명을 완성하였다.The inventors of the present invention have succeeded in separating the microorganisms in the intestines of the brown duck larvae from the brown duck larvae under the assumption that the polystyrene degrading ability is due to the microorganisms inhabiting the brown duck larvae, And thus the present invention has been completed.
상기 목적을 달성하기 위하여, 본 발명의 일 예는 갈색거저리(Tenebrio molitor) 애벌레의 장내에서 분리되고, 클레브시엘라 옥시토카(Klebsiella oxytoca), 에스케리치아 페르구소니(Escherichia fergusonii) 또는 바실러스 토요넨시스(Bacillus toyonensis) reborn(수탁번호 : KACC 81043BP)에서 선택되며, 플라스틱 분해 활성을 갖는 것을 특징으로 하는 미생물을 제공한다. 상기 갈색거저리(Tenebrio molitor) 애벌레의 장내에서 분리된 미생물은 PET(Polyethylene terephthalate), PVC(Polyvinyl chloride), PS(Polystyrene), PP(Polypropylene) 또는 PE(Polyethylene)에서 선택되는 1종 이상의 플라스틱에 대해 분해 활성을 갖는다. 또한, 상기 갈색거저리(Tenebrio molitor) 애벌레의 장내에서 분리된 미생물의 플라스틱 분해 활성은 배양 온도, 배양 시간 등에 의해 다양한 범위를 가질 수 있다. 구체적으로, 상기 갈색거저리(Tenebrio molitor) 애벌레의 장내에서 분리된 클레브시엘라 옥시토카(Klebsiella oxytoca)는 약 15일 동안 플라스틱과 함께 배양될 때 PET(Polyethylene terephthalate), PS(Polystyrene), PE(Polyethylene)에 대해 상대적으로 높은 분해 활성을 보인다. 또한, 상기 갈색거저리(Tenebrio molitor) 애벌레의 장내에서 분리된 클레브시엘라 옥시토카(Klebsiella oxytoca)는 약 30일 동안 플라스틱과 함께 배양될 때 PET(Polyethylene terephthalate), PVC(Polyvinyl chloride) 및 PP(Polypropylene)에 대해 상대적으로 높은 분해 활성을 보인다. 또한, 상기 갈색거저리(Tenebrio molitor) 애벌레의 장내에서 분리된 에스케리치아 페르구소니(Escherichia fergusonii)는 약 15일 동안 플라스틱과 함께 배양될 때 PET(Polyethylene terephthalate), PVC(Polyvinyl chloride), PS(Polystyrene), PP(Polypropylene) 및 PE(Polyethylene) 전체에 대해 높은 분해 활성을 보인다. 또한, 상기 갈색거저리(Tenebrio molitor) 애벌레의 장내에서 분리된 에스케리치아 페르구소니(Escherichia fergusonii)는 약 30일 동안 플라스틱과 함께 배양될 때 PET(Polyethylene terephthalate), PS(Polystyrene) 및 PE(Polyethylene)에 대해 상대적으로 높은 분해 활성을 보인다. 또한, 상기 갈색거저리(Tenebrio molitor) 애벌레의 장내에서 분리된 바실러스 토요넨시스(Bacillus toyonensis) reborn(수탁번호 : KACC 81043BP)은 약 15일 동안 플라스틱과 함께 배양될 때 PET(Polyethylene terephthalate), PVC(Polyvinyl chloride) 및 PE(Polyethylene)에 대해 상대적으로 높은 분해 활성을 보인다. 또한, 상기 갈색거저리(Tenebrio molitor) 애벌레의 장내에서 분리된 바실러스 토요넨시스(Bacillus toyonensis) reborn(수탁번호 : KACC 81043BP)은 약 30일 동안 플라스틱과 함께 배양될 때 PET(Polyethylene terephthalate), PVC(Polyvinyl chloride), PS(Polystyrene) 및 PE(Polyethylene)에 대해 상대적으로 높은 분해 활성을 보인다. 본 발명의 일 예에 따른 플라스틱 분해 활성을 갖는 미생물들, 배양 온도 및 배양 시간을 공정 파라미터로 조합하여 폐플라스틱 또는 플라스틱 함유 폐기물을 처리하면 특정 플라스틱을 상대적으로 많이 분해시키거나 모든 플라스틱을 재사용이 가능한 저분자 물질로 분해시킬 수 있다.In order to achieve the above object, an example of the present invention is isolated in the intestine of a Tenebrio molitor larvae and is isolated from the intestines of Klebsiella oxytoca , Escherichia sp. fergusonii ) or Bacillus toyonensis reborn (Accession No .: KACC 81043BP), and has a plastics-degrading activity. The microorganisms isolated from the intestines of the brown guppies ( Tenebrio molitor ) larvae can be used for at least one kind of plastic selected from polyethylene terephthalate (PET), polyvinyl chloride (PVC), polystyrene (PS), polypropylene (PP) Decomposition activity. In addition, the brown gill ( Tenebrio The decomposition activity of the microorganisms isolated from the intestines of the larvae may vary depending on the culture temperature, culture time, and the like. Specifically, the brown gill ( Tenebrio molitor) isolated from the gut of the larvae Klebsiella oxy cytokine (Klebsiella oxytoca) is relatively high decomposition with respect to PET (Polyethylene terephthalate), PS ( Polystyrene), PE (Polyethylene) , when incubated with plastic for 15 days Lt; / RTI > In addition, Klebsiella oxytoca isolated from the intestines of the brown tiger ( Tenebrio molitor ) larvae has been found to be effective for the production of polyethylene terephthalate (PET), polyvinyl chloride (PVC) and PP Polypropylene). In addition, the brown gill ( Tenebrio molitor ) Escherichia , separated from the intestinal tract of the larvae ( Escherichia fergusonii exhibits a high degrading activity against all of polyethylene terephthalate (PET), polyvinyl chloride (PVC), polystyrene (PS), polypropylene (PP) and polyethylene (PE) when cultured with plastic for about 15 days. In addition, the mealworm (Tenebrio molitor) isolated from the gut of the larvae Escherichia FER obtain Sony (Escherichia fergusonii exhibits a relatively high degradation activity for PET (polyethylene terephthalate), PS (polystyrene) and PE (polyethylene) when cultured with plastic for about 30 days. In addition, the brown gill ( Tenebrio molitor) isolated from the gut of the larvae Bacillus Toyo norbornene sheath (Bacillus toyonensis) reborn (accession number: KACC 81043BP) is when incubated with plastic for 15 days PET (Polyethylene terephthalate), PVC ( Polyvinyl chloride) and PE (Polyethylene Lt; RTI ID = 0.0 > decolorizing < / RTI > activity. Also, Bacillus toyonensis reborn (Accession No. KACC 81043BP) isolated from the intestines of the brown gill ( Tenebrio molitor ) larvae has been found to contain polyethylene terephthalate (PET) Poly (vinyl chloride), PS (polystyrene) and PE (polyethylene). By treating waste plastic or plastic-containing wastes by combining the microorganisms having the decomposing activity of plastics according to an embodiment of the present invention, the incubation temperature and the incubation time with process parameters, it is possible to decompose a specific plastic relatively, It can be decomposed into a low molecular substance.
상기 목적을 달성하기 위하여, 본 발명의 일 예는 플라스틱 함유 폐기물 또는 폐 플라스틱을 플라스틱 분해 활성을 갖는 미생물과 함께 배양하는 단계를 포함하는 방법으로서, 상기 플라스틱 분해 활성을 갖는 미생물은 갈색거저리(Tenebrio molitor) 애벌레의 장내에서 분리되고, 클레브시엘라 옥시토카(Klebsiella oxytoca), 에스케리치아 페르구소니(Escherichia fergusonii) 또는 바실러스 토요넨시스(Bacillus toyonensis) reborn(수탁번호 : KACC 81043BP)에서 선택되는 것을 특징으로 하는 플라스틱 분해 방법을 제공한다. 본 발명의 일 예에 따른 플라스틱 분해 방법에서 상기 플라스틱 또는 폐 플라스틱은 PET(Polyethylene terephthalate), PVC(Polyvinyl chloride), PS(Polystyrene), PP(Polypropylene) 또는 PE(Polyethylene)에서 선택되는 1종 이상으로 구성될 수 있다. 또한, 본 발명의 일 예에 따른 플라스틱 분해 방법에서 상기 미생물의 배양 온도는 25~40℃이고 배양 시간은 10~60일인 것이 바람직하나, 반드시 여기에 제한되는 것은 아니며 분해하고자 하는 플라스틱의 종류, 플라스틱 분해 활성을 갖는 미생물의 조합 등에 의해 다양한 범위에서 변경될 수 있다. 예를 들어, 본 발명의 일 예에 따른 플라스틱 분해 방법에서 상기 미생물의 배양 온도는 27~38℃에서 선택되고 배양 시간은 15~30일에서 선택될 수 있다.In order to achieve the above object, an embodiment of the present invention is a method comprising the step of cultivating a plastic-containing waste or a waste plastic together with a microorganism having a decomposition activity of plastics, wherein the microorganism having the plastic decomposition activity is Tenebrio molitor ) ≪ / RTI > in the larval's intestines, and Klebsiella oxytoca), Escherichia Pere old Sony (Escherichia fergusonii ) or Bacillus toyonensis reborn (accession number: KACC 81043BP). In the plastic decomposition method according to an exemplary embodiment of the present invention, the plastic or waste plastic may be at least one selected from the group consisting of polyethylene terephthalate (PET), polyvinyl chloride (PVC), polystyrene (PS), polypropylene (PP) Lt; / RTI > In the method for decomposing plastics according to an exemplary embodiment of the present invention, the temperature of the microorganism is preferably 25 to 40 ° C. and the incubation time is preferably 10 to 60 days. However, the present invention is not limited thereto, A combination of microorganisms having degradation activity, and the like. For example, in the method for decomposing plastics according to an embodiment of the present invention, the temperature for culturing the microorganism may be selected at 27 to 38 ° C and the incubation time may be selected at 15 to 30 days.
상기 목적을 달성하기 위하여, 본 발명의 다른 예는 플라스틱 함유 폐기물 또는 폐 플라스틱을 플라스틱 분해 활성을 갖는 효소와 반응시키는 단계를 포함하는 방법으로서, 상기 플라스틱 분해 활성을 갖는 효소는 미생물은 갈색거저리(Tenebrio molitor) 애벌레의 장내에서 분리되고, 클레브시엘라 옥시토카(Klebsiella oxytoca), 에스케리치아 페르구소니(Escherichia fergusonii) 또는 바실러스 토요넨시스(Bacillus toyonensis) reborn(수탁번호 : KACC 81043BP)에서 선택되는 미생물이 생산하는 것을 특징으로 하는 플라스틱 분해 방법을 제공한다. 본 발명의 다른 예에 따른 플라스틱 분해 방법에서 상기 플라스틱 또는 폐 플라스틱은 PET(Polyethylene terephthalate), PVC(Polyvinyl chloride), PS(Polystyrene), PP(Polypropylene) 또는 PE(Polyethylene)에서 선택되는 1종 이상으로 구성될 수 있다. 본 발명의 다른 예에 따른 플라스틱 분해 방법에서 상기 상기 플라스틱 분해 활성을 갖는 효소는 플라스틱 분해 활성을 갖는 미생물의 세포 외 물질 또는 세포내 물질일 수 있고, 다양한 유전자 재조합을 통해 대량으로 생산할 수 있다.In order to achieve the above object, another embodiment of the present invention is a method comprising the step of reacting a plastic-containing waste or a waste plastic with an enzyme having a decomposition activity of plastics, wherein the enzyme having the plastic decomposition activity is a microorganism belonging to Tenebrio molitor ) is isolated from the larvae intestinal tract, and Klebsiella oxytoca , Escherichia wherein the microorganism is selected from the group consisting of fergusonii or Bacillus toyonensis reborn (accession number: KACC 81043BP). In the plastic decomposition method according to another embodiment of the present invention, the plastic or waste plastic may be at least one selected from the group consisting of polyethylene terephthalate (PET), polyvinyl chloride (PVC), polystyrene (PS), polypropylene (PP) Lt; / RTI > In the plastic decomposition method according to another embodiment of the present invention, the enzyme having the plastic decomposition activity may be an extracellular substance or an intracellular substance of a microorganism having a plastic decomposing activity, and can be mass-produced through various gene recombination.
본 발명에 따른 플라스틱 분해 방법은 플라스틱 재활용을 위한 전처리 과정으로 적용될 수 있다.The plastic decomposition method according to the present invention can be applied as a pretreatment process for plastic recycling.
본 발명에 따른 플라스틱 분해 활성을 갖는 미생물은 PET(Polyethylene terephthalate), PVC(Polyvinyl chloride), PS(Polystyrene), PP(Polypropylene) 또는 PE(Polyethylene)에서 선택되는 1종 이상의 플라스틱을 적정 배양 조건에서 선택적으로 또는 전체적으로 분해하여 저분자 물질로 전환시킬 수 있다. 따라서, 본 발명에 따른 플라스틱 분해 활성을 갖는 미생물은 플라스틱 재활용을 위한 전처리 과정에 사용될 수 있다.The microorganism having the decomposition activity of plastics according to the present invention may be selected from at least one plastic selected from polyethylene terephthalate (PET), polyvinyl chloride (PVC), polystyrene (PS), polypropylene (PP) Or can be converted into a low-molecular substance by decomposition as a whole. Therefore, the microorganism having the decomposing activity of plastics according to the present invention can be used in a pretreatment process for plastic recycling.
도 1은 고체 배지를 이용한 플라스틱 분해 실험에서 갈색거저리 애벌레의 장내에서 분리된 28℃-2 미생물의 플라스틱 분해 활성을 나타낸 것이다.
도 2는 고체 배지를 이용한 플라스틱 분해 실험에서 갈색거저리 애벌레의 장내에서 분리된 28℃-2-1 미생물의 플라스틱 분해 활성을 나타낸 것이다.
도 3은 고체 배지를 이용한 플라스틱 분해 실험에서 갈색거저리 애벌레의 장내에서 분리된 28℃-3-1 미생물의 플라스틱 분해 활성을 나타낸 것이다.
도 4는 고체 배지를 이용한 플라스틱 분해 실험에서 갈색거저리 애벌레의 장내에서 분리된 28℃-8 미생물의 플라스틱 분해 활성을 나타낸 것이다.
도 5는 고체 배지를 이용한 플라스틱 분해 실험에서 갈색거저리 애벌레의 장내에서 분리된 37℃-1 미생물의 플라스틱 분해 활성을 나타낸 것이다.
도 6은 고체 배지를 이용한 플라스틱 분해 실험에서 갈색거저리 애벌레의 장내에서 분리된 37℃-2 미생물의 플라스틱 분해 활성을 나타낸 것이다.
도 7은 고체 배지를 이용한 플라스틱 분해 실험에서 갈색거저리 애벌레의 장내에서 분리된 37℃-2-1 미생물의 플라스틱 분해 활성을 나타낸 것이다.
도 8은 고체 배지를 이용한 플라스틱 분해 실험에서 갈색거저리 애벌레의 장내에서 분리된 37℃-6 미생물의 플라스틱 분해 활성을 나타낸 것이다.
도 9는 고체 배지를 이용한 플라스틱 분해 실험에서 갈색거저리 애벌레의 장내에서 분리된 37℃-7 미생물의 플라스틱 분해 활성을 나타낸 것이다.
도 10은 고체 배지를 이용한 플라스틱 분해 실험에서 갈색거저리 애벌레의 장내에서 분리된 37℃-9 미생물의 플라스틱 분해 활성을 나타낸 것이다.
도 1 내지 도 10에서 왼쪽의 플레이트는 플라스틱 필름을 설치한 실험군이고 오른쪽의 플레이트는 플라스틱 필름을 설치하지 않은 대조군이다.
도 11은 액체 배지를 이용한 PVC(Polyvinyl chloride) 분해 실험에서 배양 15일 후 대조군(Control)의 배양액을 가스크로마토그래피로 분석한 결과이다.
도 12는 액체 배지를 이용한 PVC(Polyvinyl chloride) 분해 실험에서 배양 15일 후 선별된 균주 에스케리치아 페르구소니(Escherichia fergusonii)의 배양액을 가스크로마토그래피로 분석한 결과이다.FIG. 1 shows the decomposition activity of 28 ° C. -2 microorganisms isolated from the intestine of a brown dung larvae in a plastic decomposition experiment using a solid medium.
Fig. 2 shows plastics degradation activity of 28 ° C-2-1 microorganisms isolated from the intestines of brown duck larvae in a plastic decomposition experiment using a solid medium.
Fig. 3 shows the decomposition activity of the 28 ° C-3-1 microorganism isolated from the intestines of the brown dung larvae in the plastic decomposition experiment using the solid medium.
Fig. 4 shows plastics degradation activity of 28 ° C-8 microorganisms isolated from the intestines of brown duck larvae in a plastic decomposition experiment using a solid medium.
Fig. 5 shows the decomposition activity of the 37 ° C-1 microorganism isolated from the intestines of the brown duck larvae in the plastic decomposition experiment using the solid medium.
FIG. 6 shows plastics degradation activity of the 37 ° C. -2 microorganism isolated from the intestines of brown gill larvae in a plastic decomposition experiment using a solid medium.
FIG. 7 shows the decomposition activity of the 37 ° C. -2-1 microorganism isolated from the intestines of the brown duck larvae in the plastic decomposition experiment using the solid medium.
FIG. 8 shows plastics degradation activity of 37 ° C. -6 microorganisms isolated from the intestines of brown goose larvae in a plastic decomposition experiment using a solid medium.
Fig. 9 shows the decomposition activity of the 37 ° C-7 microorganism isolated from the intestines of the brown duck larvae in a plastic decomposition experiment using a solid medium.
10 shows the decomposition activity of the 37 ° C-9 microorganism isolated from the intestines of the brown duck larvae in a plastic decomposition experiment using a solid medium.
In FIGS. 1 to 10, the left plate is an experimental group with a plastic film, and the right plate is a control group without a plastic film.
FIG. 11 shows the result of gas chromatography analysis of the culture of control (control) 15 days after the culture in a polyvinyl chloride (PVC) decomposition experiment using a liquid medium.
12 is a PVC (Polyvinyl chloride) selected after 15 days incubation in the degradation experiments strain Escherichia FER obtain Sony (Escherichia using a liquid culture medium fergusonii ) was analyzed by gas chromatography.
이하, 본 발명을 실시예를 통하여 구체적으로 설명한다. 다만, 하기 실시예는 본 발명의 기술적 특징을 명확하게 예시하기 위한 것 일뿐, 본 발명의 보호범위를 한정하는 것은 아니다.Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are intended to clearly illustrate the technical features of the present invention and do not limit the scope of protection of the present invention.
1. One. 갈색거저리Brown ass (( TenebrioTenebrio molitormolitor ) 애벌레의 장내 미생물 분리, 선별 및 동정Separation, selection and identification of intestinal microorganisms in larvae
(1) 갈색거저리 애벌레의 장내 미생물 분리(1) Isolation of intestinal microorganisms of brown duck larvae
2주 동안 발포폴리스티렌(Expanded polystyrene, EPS) 및 폴리비닐클로라이드(Polyvinyl chloride, PVC)만을 섭취한 갈색거저리 애벌레 약 40마리(총 무게 : 5g)를 생리식염수(0.85% NaCl) 50㎖에 투입하고 분쇄기로 약 30초 동안 분쇄하여 갈색거저리 애벌레 분쇄액을 수득하였다. 이후, 갈색거저리 애벌레 분쇄액을 생리식염수를 사용하여 다양한 배율(10-4, 10-5, 10-6, 10-7)로 희석하여 4개의 갈색거저리 애벌레 희석액을 수득하였다. 이후, 갈색거저리 애벌레 희석액을 LB 한천 배지 및 Difco 한천 배지에 접종하여 총 16개의 접종 플레이트를 제작하고 28℃ 및 37℃에서 2일 동안 배양하여 멀티 콜로니(multi colonies)가 형성된 배양 플레이트를 수득하였다. 상기 LB 한천 배지는 증류수 500㎖에 LB Broth 10g 및 한천(Agar) 10g을 첨가하고 혼합한 후 멸균기에서 40분 동안 멸균한 것이다. 또한, 상기 Difco 한천 배지는 증류수 500㎖에 Difco nutrient broth 1.5g, Yeast extract 12.5g 및 한천(Agar) 10g을 첨가하고 혼합한 후 멸균기에서 40분 동안 멸균한 것이다. 이후, 배양 플레이트에 형성된 멀티 콜로니로부터 각 콜로니를 취하고 LB 한천 배지 및 Difco 한천 배지에 도말(streaking)한 후 대응되는 멀티 콜로니 배양 조건과 동일한 조건으로 1일 동안 배양하여 총 28종의 단일 콜로니를 수득하였고, 색상, 형상의 매끈함 정도 및 꼬리 운동성을 기준으로 재분류하여 총 19종의 단일 콜로니를 수득하였다. 총 19종의 단일 콜로니 중 7종은 28℃에서 배양한 것이고 12종은 37℃에서 배양한 것이다.Approximately 40 brown gill larvae (total weight: 5 g) consumed only expanded polystyrene (EPS) and polyvinyl chloride (PVC) for 2 weeks were placed in 50 ml of physiological saline (0.85% NaCl) For about 30 seconds to obtain a brown gruel larvae pulverization liquid. Thereafter, the brown shag mite larvae were diluted with physiological saline to various magnifications (10 -4 , 10 -5 , 10 -6 , 10 -7 ) to obtain four brown gill larva dilutions. Then, a total of 16 inoculation plates were prepared by inoculating a brown duck larvae diluent into LB agar medium and Difco agar medium, and cultured at 28 ° C and 37 ° C for 2 days to obtain a culture plate in which multi colonies were formed. The LB agar medium was prepared by adding 10 g of LB broth and 10 g of agar to 500 ml of distilled water, mixing and sterilizing for 40 minutes in a sterilizer. The Difco agar medium was prepared by adding 1.5 g of Difco nutrient broth, 12.5 g of yeast extract and 10 g of agar to 500 ml of distilled water, mixing and sterilizing for 40 minutes in a sterilizer. Then, each colony was taken from a multi-colony formed on a culture plate, streaked on an LB agar medium and a Difco agar medium, and cultured for one day under the same conditions as the corresponding multicolumn culture conditions to obtain a total of 28 single colonies , And reclassified on the basis of the degree of smoothness and tail motility of color, shape, and 19 kinds of single colonies were obtained. Seven of the 19 single colonies were cultured at 28 ℃ and 12 were cultured at 37 ℃.
(2) 갈색거저리 애벌레의 장내에서 분리된 미생물의 플라스틱 분해 활성 측정(고체 배지)(2) Determination of the decomposing activity of the microorganisms isolated from the intestines of brown duck larvae (solid medium)
앞에서 수득한 총 19종의 단일 콜로니를 탄소원이 없는 기본 한천 배지(Carbon-free basal agar medium, CFBAM) 상에 각각 도말(streaking)하여 CFBAM 플레이트를 제작하였다. 상기 CFBAM은 증류수 1000㎖에 KH2PO4 0.7g, K2HPO4 0.7g, MgSO4·H2O 0.7g, NH4NO3 1.0g, NaCl 0.005g, FeSO4·H2O 0.002g, ZnSO4·H2O 0.002g, MnSO4·H2O 0.001g 및 한천(agar) 15g을 첨가하고 혼합한 후 멸균기에서 40분 동안 멸균한 것이다. 이후, CFBAM 플레이트를 4개의 영역으로 분할하고 각 영역에 폴리스티렌(Polystyrene, PS) 필름, (Polyvinyl chloride, PVC) 필름, 폴리프로필렌(Polypropylene, PP) 필름 및 폴리에틸렌테레프탈레이트(Polyethylene terephthalate, PET) 필름을 설치하였다. CFBAM은 탄소원이 없기 때문에 미생물을 플라스틱 필름을 분해하지 않으면 생존할 수 있다. 다만, 매우 희박하게 생존을 위해 한천(agar)을 분해하는 미생물이 존재할 수 있으므로, 대조군(Control group)으로 CFBAM 상에 단일 콜로니를 각각 도말하고 별도의 플라스틱 필름을 설치하지 않은 CFBAM 플레이트를 제작하였다. 이후, 플라스틱 필름이 설치된 CFBAM 플레이트 및 플라스틱 필름이 설치되지 않은 CFBAM 플레이트를 28℃ 또는 37℃에서 28일 동안 배양하였다. 플라스틱 필름의 주변에서 성장하면서 동시에 대응되는 대조군에서 성장하지 않는 미생물은 플라스틱 분해 활성을 갖는 것으로 여겨질 수 있다. 28일 동안의 배양 후에 10종의 미생물이 필름 가장자리에서 생존하였고, 구체적인 결과는 하기 표 1 및 도 1 내지 도 10과 같다.CFBAM plates were prepared by streaking each of the 19 single colonies obtained in the above on a carbon-free basal agar medium (CFBAM). The CFBAM is in distilled
(배양 온도-No.)Microbiological classification
(Incubation temperature-No.)
상기 표 1에서 '○'는 플라스틱 필름 주변에 미생물이 자랐다는 것을 의미하고, '×'는 플라스틱 필름 주변에 미생물이 자라지 않았다는 것을 의미한다. 한편, 28℃-2-1 미생물의 경우 대조군에서 미약한 형태로 아주 적은 양이 자란 반면, 해당 플라스틱 필름 주변에서 밀집된 형태로 아주 많은 양이 자랐기 때문에 플라스틱 분해 활성을 갖는 미생물에 포함하였다.In Table 1, "o" means that microorganisms were grown around the plastic film, and "x" means that microorganisms did not grow around the plastic film. On the other hand, 28 ° C -2-1 microorganisms were included in the microorganisms having plastic decomposition activity because they were grown in a very small amount in a dense form around the plastic film while they were grown in a very small amount in the control group.
(3) 미생물의 동정 및 선별(3) Identification and selection of microorganisms
상기 표 1에 기재된 미생물의 DNA를 추출하고 증폭한 후 16S rDNA 서열을 분석하여 미생물의 종을 확정하였다. 상기 표 1에 기재된 10종의 미생물 중 병원성이 매우 강한 미생물과 플라스틱 분해 스펙트럼이 작은 미생물을 제외하고 28℃-2-1 미생물, 28℃-3-1 미생물 및 37℃-2-1 미생물을 최종 균주로 선별하였다. 하기 표 2에 선별된 3종의 균주에 대한 종명 및 선별된 3종의 균주와 유사한 16S rDNA 서열을 갖는 공지의 균주를 나타내었다.The DNA of the microorganism described in Table 1 was extracted and amplified, and the 16S rDNA sequence was analyzed to confirm the species of the microorganism. Among the 10 microorganisms listed in Table 1, except for microorganisms having extremely high pathogenicity and microorganisms having a small plastic decomposition spectrum, 28 ° C -2-1 microorganisms, 28 ° C -3-1 microorganisms and 37 ° C -2-1 microorganisms . The strains were selected for the three strains selected in Table 2 below, and a known strain having the 16S rDNA sequence similar to the selected three strains was shown.
(배양 온도-No.)Microbiological classification
(Incubation temperature-No.)
2. 선별된 미생물의 액체 배지 상에서의 플라스틱 분해 활성(액상 배지)2. Plastics degradation activity of the selected microorganisms on the liquid medium (liquid medium)
폐 플라스틱 재활용 공정 중 위에서 선별된 미생물 또는 선별된 미생물이 생산하는 효소로 일부 플라스틱을 분해하는 전처리 과정은 통상적으로 액상 조건에서 행해진다. 액상 배지를 이용하여 폐 플라스틱 재활용 공정 중의 전처리 과정과 유사한 조건에서 선별된 미생물의 플라스틱 분해 활성을 측정하였다. 구체적으로, 증류수 1000㎖에 KH2PO4 0.7g, K2HPO4 0.7g, MgSO4·H2O 0.7g, NH4NO3 1.0g, NaCl 0.005g, FeSO4·H2O 0.002g, ZnSO4·H2O 0.002g, 및 MnSO4·H2O 0.001g을 첨가하고 혼합한 후 멸균기로 40분 동안 멸균하여 탄소원이 없는 액상 기본 배지(Liquid carbon-free basal medium, LCFBM)을 제조하였다. 이후, 삼각 플라스크에 LCFBM 40㎖와 플라스틱 입자(PET, PVC, PS, PP, PE) 100㎎을 각각 넣고, 여기에 선별된 미생물 3종을 각각 108 CFU/㎖의 농도로 접종하고, 진탕 배양기를 이용하여 28℃에서 30일 동안 배양하였다. 또한, 삼각 플라스크에 LCFBM 및 플라스틱 입자만을 각각 넣고 진탕 배양기를 이용하여 28℃에서 30일 동안 배양한 후, 이를 대조군(Control)으로 사용하였다.The pretreatment process for decomposing some plastics with enzymes produced by selected microorganisms or selected microorganisms in the waste plastic recycling process is usually carried out under liquid conditions. The microbial degradation activity of the selected microorganisms was measured under similar conditions to the pretreatment process in waste plastic recycling process using liquid medium. Specifically, in distilled
미생물 배양 후 15일 및 30일이 되는 날 배양액을 샘플링하고 플라스틱 입자의 중량 감소를 측정하였다. 하기 표 3은 액체 배지를 이용한 플라스틱 분해 실험에서, 미생물 배양 후 15일이 되었을 때의 플라스틱 입자 감소량을 나타낸 것이고, 하기 표 4는 액체 배지를 이용한 플라스틱 분해 실험에서, 미생물 배양 후 30일이 되었을 때의 플라스틱 입자 감소량을 나타낸 것이다. 하기 표 3에서 보이는 바와 같이 미생물 배양 후 15일이 되었을 때 선별된 균주 클레브시엘라 옥시토카(Klebsiella oxytoca)는 PET, PS 및 PP에 대해 분해 활성이 높았고, 선별된 균주 에스케리치아 페르구소니(Escherichia fergusonii)는 PET, PVC, PS, PP 및 PE 모두에 대해 분해 활성이 높았고, 선별된 균주 바실러스 토요넨시스(Bacillus toyonensis)는 PET, PVC, PS 및 PE에 대해 분해 활성이 높았다. 또한, 하기 표 4에서 보이는 바와 같이 미생물 배양 후 30일이 되었을 때 선별된 균주 클레브시엘라 옥시토카(Klebsiella oxytoca)는 PET, PVC 및 PP에 대해 상대적으로 분해 활성이 높았고, 선별된 균주 에스케리치아 페르구소니(Escherichia fergusonii)는 PET, PS 및 PP에 대해 상대적으로 분해 활성이 높았고, 선별된 균주 바실러스 토요넨시스(Bacillus toyonensis)는 PET, PVC, PS 및 PE에 대해 상대적으로 분해활성이 높았다.15 days and 30 days after the microbial culturing, the culture fluid of the blades was sampled and the weight loss of the plastic particles was measured. Table 3 below shows the amount of plastic particles decreased at the 15th day after the microbial cultivation in the plastic decomposition experiment using the liquid medium. Table 4 below shows the results of the plastic decomposition experiment using the liquid medium, Of the amount of plastic particles. As shown in Table 3, Klebsiella oxytoca ( Klebsiella oxytoca ), which was selected at the 15th day after culturing the microorganism, showed a high degrading activity against PET, PS and PP, and the selected strain Escherichia pergoni Escherichia fergusonii showed high degradative activity against both PET, PVC, PS, PP and PE. The selected strain Bacillus toyonensis showed high degradation activity against PET, PVC, PS and PE. As shown in Table 4, Klebsiella oxytoca (strain Klebsiella oxytoca ), which was selected at 30 days after the cultivation of the microorganism, had a relatively high decomposition activity relative to PET, PVC and PP, and the selected strain Escherichia coli Escherichia fergusonii ) showed higher degradation activity relative to PET, PS and PP, and the selected strain Bacillus toyonensis had a higher degradation activity relative to PET, PVC, PS and PE.
* PET : Polyethylene terephthalate* PET: Polyethylene terephthalate
* PVC : Polyvinyl chloride* PVC: Polyvinyl chloride
* PS : Polystyrene* PS: Polystyrene
* PP : Polypropylene* PP: Polypropylene
* PE : Polyethylene* PE: Polyethylene
또한, 미생물 배양 후 15일이 되었을 때 샘플링한 배양액을 여과하고 여과액을 가스크로마토그래피로 분석하여 저분자 물질의 증가 여부를 확인하였다. 가스크로마토그래피 분석 결과 대부분의 배양액에서 저분자 물질의 함량이 크게 증가하였다. 도 11은 액체 배지를 이용한 PVC(Polyvinyl chloride) 분해 실험에서 배양 15일 후 대조군(Control)의 배양액을 가스크로마토그래피로 분석한 결과이다. 도 12는 액체 배지를 이용한 PVC(Polyvinyl chloride) 분해 실험에서 배양 15일 후 선별된 균주 에스케리치아 페르구소니(Escherichia fergusonii)의 배양액을 가스크로마토그래피로 분석한 결과이다. 도 11 및 도 12에서 보이는 바와 같이 에스케리치아 페르구소니(Escherichia fergusonii)의 배양액에서 체류 시간(retention time)이 짧은 저분자 물질의 피크 면적이 대조군(Control)의 배양액에 비해 크게 증가하였다.In addition, at 15 days after the microbial culture, the sampled culture was filtered, and the filtrate was analyzed by gas chromatography to ascertain whether the amount of the low molecular substance was increased. Gas chromatographic analysis showed that the content of low molecular weight substances in most of the culture media was greatly increased. FIG. 11 shows the result of gas chromatography analysis of the culture of control (control) 15 days after the culture in a polyvinyl chloride (PVC) decomposition experiment using a liquid medium. 12 is a PVC (Polyvinyl chloride) selected after 15 days incubation in the degradation experiments strain Escherichia FER obtain Sony (Escherichia using a liquid culture medium fergusonii ) was analyzed by gas chromatography. As shown in Figures 11 and 12, Escherichia < RTI ID = 0.0 > fergusonii ), the peak area of the low molecular weight substance with short retention time was significantly increased compared to the control culture.
3. 선별된 미생물의 3. Selection of microorganisms 균주명 부여Strain naming 및 수탁 And trust
본 발명의 발명자들은 선별된 37℃-2-1 미생물에게 바실러스 토요넨시스(Bacillus toyonensis) reborn 이라는 균주명을 부여하였다. 또한, 본 발명의 발명자들은 바실러스 토요넨시스(Bacillus toyonensis) reborn을 2017년 1월 17일에 공인기탁기관인 국립농업과학원(주소 : 대한민국 전북 완주군 이서면 농생명로 166)에 특허기탁 하여 KACC 81043BP의 수탁번호를 부여받았다. 상기 미생물의 기탁은 특허 절차상의 미생물기탁의 국제적 승인에 관한 부다페스트조약(Budapest Treaty on the International Recognition of the Deposit of Microorganism for the Purposes of Patent Procedure)을 준수하여 이루어졌다.The inventors of the present invention gave a strain name of Bacillus toyonensis reborn to the selected 37 ° C -2-1 microorganism. The inventors of the present invention also deposited a patent on Bacillus toyonensis reborn on January 17, 2017 to the National Institute of Agricultural Science and Technology (National Institute of Agricultural Science and Technology, 166, Jeonbuk, Jeonbuk, Republic of Korea) . The deposit of such microorganisms has been made in accordance with the Budapest Treaty on the Recognition of Microorganisms for the Purposes of Patent Procedure on the International Recognition of Microorganism Deposit under the Patent Procedure.
이상에서와 같이 본 발명을 상기의 실시예를 통해 설명하였지만 본 발명이 반드시 여기에만 한정되는 것은 아니며 본 발명의 범주와 사상을 벗어나지 않는 범위 내에서 다양한 변형실시가 가능함은 물론이다. 따라서, 본 발명의 보호범위는 본 발명에 첨부된 특허청구의 범위에 속하는 모든 실시 형태를 포함하는 것으로 해석되어야 한다.While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. Therefore, the scope of the present invention should be construed as including all embodiments falling within the scope of the appended claims.
Claims (7)
클레브시엘라 옥시토카(Klebsiella oxytoca), 에스케리치아 페르구소니(Escherichia fergusonii) 또는 바실러스 토요넨시스(Bacillus toyonensis) reborn(수탁번호 : KACC 81043BP)에서 선택되며,
플라스틱 분해 활성을 갖는 것을 특징으로 하는 미생물.
Brown Tortoise ( Tenebrio molitor ) separated from the intestines of the larvae,
Klebsiella oxytoca , Escherichia fergusonii , or Bacillus toyonensis reborn (accession number: KACC 81043BP)
A microorganism having a plastic decomposition activity.
The microorganism according to claim 1, wherein the plastic is at least one selected from the group consisting of polyethylene terephthalate (PET), polyvinyl chloride (PVC), polystyrene (PS), polypropylene (PP)
상기 플라스틱 분해 활성을 갖는 미생물은 갈색거저리(Tenebrio molitor) 애벌레의 장내에서 분리되고, 클레브시엘라 옥시토카(Klebsiella oxytoca), 에스케리치아 페르구소니(Escherichia fergusonii) 또는 바실러스 토요넨시스(Bacillus toyonensis) reborn(수탁번호 : KACC 81043BP)에서 선택되는 것을 특징으로 하는 플라스틱 분해 방법.
A method comprising the step of culturing a plastic-containing waste or a waste plastic together with a microorganism having a plastic decomposition activity,
The microorganism having the above-mentioned plastic decomposing activity was a brown gruel ( Tenebrio molitor ) is isolated from the larvae intestinal tract, and Klebsiella oxytoca), Escherichia Pere old Sony (Escherichia fergusonii ) or Bacillus toyonensis reborn (accession number: KACC 81043BP).
The method of claim 1, wherein the plastic or waste plastic is at least one selected from the group consisting of polyethylene terephthalate (PET), polyvinyl chloride (PVC), polystyrene (PS), polypropylene (PP) Way.
The plastic decomposition method according to claim 4, wherein the culture temperature of the microorganism is 25 to 40 ° C. and the culture time is 10 to 60 days.
상기 플라스틱 분해 활성을 갖는 효소는 미생물은 갈색거저리(Tenebrio molitor) 애벌레의 장내에서 분리되고, 클레브시엘라 옥시토카(Klebsiella oxytoca), 에스케리치아 페르구소니(Escherichia fergusonii) 또는 바실러스 토요넨시스(Bacillus toyonensis) reborn(수탁번호 : KACC 81043BP)에서 선택되는 미생물이 생산하는 것을 특징으로 하는 플라스틱 분해 방법.
A method comprising reacting a plastic-containing waste or a waste plastic with an enzyme having a plastic decomposition activity,
The enzyme having the above-mentioned plastic decomposing activity is characterized in that the microorganism is isolated in the intestine of a brown duck ( Tenebrio molitor ) larva, and Klebsiella oxytoca), Escherichia Pere old Sony (Escherichia wherein the microorganism is selected from the group consisting of fergusonii or Bacillus toyonensis reborn (accession number: KACC 81043BP).
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| PCT/KR2018/001517 WO2018143750A1 (en) | 2017-02-03 | 2018-02-05 | Microorganism isolated from tenebrio molitor larva and having plastic degrading activity, and method for degrading plastic using same |
| EP18747454.9A EP3587557A4 (en) | 2017-02-03 | 2018-02-05 | Microorganism isolated fromtenebrio |
| CN201880010250.XA CN111094543B (en) | 2017-02-03 | 2018-02-05 | Microorganism having plastic degradation activity separated from yellow meal worm larva and plastic degradation method using same |
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| CN111094543B (en) | 2023-12-22 |
| WO2018143750A1 (en) | 2018-08-09 |
| EP3587557A4 (en) | 2020-12-02 |
| EP3587557A1 (en) | 2020-01-01 |
| KR102126889B1 (en) | 2020-06-26 |
| CN111094543A (en) | 2020-05-01 |
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